Nitrogenase Activity Of Cells Research Articles

Effect of the over-expression of PII and PZ proteins on the nitrogenase activity of Azospirillum brasilense

The Azospirillum brasilense PII and PZ proteins, encoded by the glnB and glnZ genes respectively, are intracellular transducers of nitrogen levels with distinct functions. The PII protein participates in nif regulation by controlling the activity of the transcriptional regulator NifA. PII is also involved in transducing the prevailing nitrogen levels to the Fe-protein ADP-ribosylation system. PZ regulates negatively ammonium transport and is involved in nitrogenase reactivation. To further investigate the role of PII and PZ in the regulation of nitrogen fixation, broad-host-range plasmids capable of over-expressing the glnB and glnZ genes under control of the ptac promoter were constructed and introduced into A. brasilense. The nitrogenase activity and nitrate-dependent growth was impaired in A. brasilense cells over-expressing the PII protein. Using immunoblot analysis we observed that the reduction of nitrogenase activity in cells over-expressing PII was due to partial ADP-ribosylation of the Fe-protein under derepressing conditions and a reduction in the amount of Fe-protein. These results support the hypothesis that the unmodified PII protein act as a signal to the DraT enzyme to ADP-ribosylate the Fe-protein in response to ammonium shock, and that it also inhibits nif gene expression. In cells over-expressing the PZ protein the nitrogenase reactivation after an ammonium shock was delayed indicating that the PZ protein is involved in regulation of DraG activity.

Control by light of whole-cell nitrogenase activity and of nitrogenase and bacteriochlorophyll a formation in Rhodobacter capsulatus strain B10S

Control of nitrogenase and bacteriochlorophyll a (BChl) by light was studied under steady-state conditions with continuous cultures of Rhodobacter capsulatus B10S supplied with malate and growth-limiting amounts of ammonium. Consumption of malate and, correspondingly, the C/N ratio at which malate and ammonium were consumed increased when illumination was increased from 3 to approximately 20 klx and became constant at higher illuminations of up to 40 klx. Essentially the same kinetics were observed with respect to nitrogenase activity of cells, contents of nitrogenase polypeptides, and nifH promoter activity. Substrate consumption was half-maximal at 8 klx and was independent of the presence of nitrogenase. Therefore, it is concluded that light controls the C/N ratio (a quantitative measure of the nitrogen status of cells), which in turn is involved in the control of nitrogenase at the level of nif promoter activity. Post-translational regulation of nitrogenase activity by ADP-ribosylation was not observed under steady-state conditions, but it took place when illumination was suddenly decreased to the range where malate consumption and, consequently, the C/N ratio decreased. Irrespective of the presence or absence of nitrogenase, specific BChl contents of the cultures were constant above 20 klx, and they increased at lower illuminations. These results do not confirm a recently proposed link between nitrogen fixation and photosynthesis as represented by BChl.

Increased Respiration Through Cytochrome d Enhances Microaerobic N2Fixation in Klebsiella Pneumoniae

Nitrogenase activity was increased in a Klebsiella pneumoniae strain (FN27) producing higher amounts of cytochrome d than the wild-type strain. The increased production of cytochrome d in FN27 showed a positive effect on nitrogenase activity in cells cultured with glucose as carbon source at 1 kPa oxygen but a negative effect at higher O2concentrations. In cells cultured with pyruvate as carbon source, FN27 expressed higher activity of nitrogenase at all oxygen tensions tested when compared to the wild-type strain. This analysis shows that the over production of cytochrome d terminal oxidase improves nitrogen fixation in certain culture conditions.

Properties of the Peribacteroid Membrane ATPase of Pea Root Nodules and Its Effect on the Nitrogenase Activity.

Peribacteroid membrane vesicles from pea (Pisum sativum) root nodules were isolated from membrane-enclosed bacteroids by an osmotic shock. The ATPase activity associated with this membrane preparation was characterized, and its electrogenic properties were determined. The pH gradient was measured as a change of the fluorescence intensity of 9-amino-6-chloro-2-methoxyacridine and the membrane potential as a shift of absorbance of bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. It was demonstrated that the ATPase generates a pH gradient as well as a membrane potential across the peribacteroid membrane. The reversibility of the ATPase was demonstrated by a light-dependent ATP synthesis by peribacteroid membrane vesicles fused with bacteriorhodopsin-phospholipid vesicles. The light-driven ATP synthesis by the peribacteroid membrane ATPase was completely inhibited by a proton-conducting ionophore. The proton-pumping activity of the peribacteroid membrane ATPase could also be demonstrated with peribacteroid membrane-enclosed bacteroids, and effects on nitrogenase activity were established. At pH values below 7.5, an active peribacteroid membrane ATPase inhibited the nitrogenase activity of peribacteroid membrane-enclosed bacteroids. At pH values above 8, at which whole cell nitrogenase activity was inhibited, the protonpumping activity of the peribacteroid membrane ATPase could partially reverse the pH inhibition. Vanadate, an inhibitor of plasma membrane and peribacteroid membrane ATPases, stimulated nodular nitrogenase activity. It will be proposed that the proton-pumping activity of the peribacteroid membrane ATPase in situ is a possible regulator of nodular nitrogenase activity.

Adenine nucleotides, substrate utilization and dinitrogen fixation in Rhodobacter capsulatus

Rhodobacter capsulatus strain 37b4 was grown diazotrophically in phototrophic chemostat culture with 30 mM of d,l-malate and 2 mM of ammonium. Illumination was varied at constant dilution rate (D) and vice versa, respectively. When D was raised from 0.035 to 0.165 h-1 at 30 klx, the steady state cell protein level as well as malate consumption decreased. d-malate was utilized only at D=0.035 h-1. Specific cellular activities of nitrogenase, as determined by acetylene reduction as well as by dinitrogen (N2) fixation, increased and approached constancy at D>0.075 h-1. Specific ATP contents of cells increased with increasing D, while specific ADP and AMP contents exhibited no significant variations. Consequently, energy charge values as well as molar ratios of ATP/ADP (T/D) increased. Raising illumination from 6 to 30 klx at D=0.075 h-1 resulted in an increase of the steady state protein level as well as of l-malate consumption. d-malate was not utilized under these conditions. Specific nitrogenase activity of cells increased at the lower and levelled off at the higher illuminations. Specific ATP contents of cells stayed constant but specific ADP contents increased with increasing illumination. The energy charge did not vary significantly, while the T/C ratio decreased between 6 and 18 klx and stayed constant at the higher illuminations. The results do not reveal any relationship between nitrogenase activity and the cellular levels or relative proportions of different adenine nucleotides. However, when steady state amounts of fixed N2 were plotted versus steady state T/D ratios, an inverse proportion became apparent, irrespective of the growth conditions employed. On the other hand, specific nitrogenase activity increased linearly when the rate of malate consumption increased. The results suggest that under steady state conditions the T/D ratio reflects the amount of ATP required to keep the amount of fixed N2 at a given level, while the rate at which nitrogenase functions depends on the rate at which the carbon and electron source, malate, is utilized by the organisms.

Regulation of nitrogen-fixation by different nitrogen sources in the marine non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067

The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N2 as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N2 was not suppressed after 7 h incubation with 2 mM NaNO3 or 0.02 mM NH4Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO3, 0.5 mM urea or 0.02 mM NH4Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo−Fe protein, revealed the following: (1) both the Fe-protein and the Mo−Fe protein were synthesized in cells grown with N2 as well as in cells grown with NaNO3 or low concentration of NH4Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO3 or low concentration of NH4Cl; (4) neither the Fe-protein nor the Mo−Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1.

Catabolic repression of hydrogenase synthesis in Ectothiorhodospira shaposhnikovii

The hydrogenase activity of purple sulfur bacterium Ectothiorhodospira shaposhnikovii was minimal when grown in acetate excess. Molecular hydrogen evolved to the nitrogenase activity of cells cultivated in chemostat under ammonium limitation did not increase the hydrogenase activity. Under acetate limitation, the flow rate decline increased the hydrogenase activity of cells and simultaneously the intracellular content of cyclic adenosine-5′-monophospate. Therefore, the hydrogenase synthesis by E. shaposhnikovii is regulated by catabolic repression.

Catabolic repression of hydrogenase synthesis in Ectothiorhodospira shaposhnikovii

The hydrogenase activity of purple sulfur bacterium Ectothiorhodospira shaposhnikovii was minimal when grown in acetate excess. Molecular hydrogen evolved to the nitrogenase activity of cells cultivated in chemostat under ammonium limitation did not increase the hydrogenase activity. Under acetate limitation, the flow rate decline increased the hydrogenase activity of cells and simultaneously the intracellular content of cyclic adenosine-5′-monophospate. Therefore, the hydrogenase synthesis by E. shaposhnikovii is regulated by catabolic repression.

Regulation of nitrogen fixation by nitrite and glutamine in Derxia gummosa

Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C2H2 reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C2H2 reduction had a Ki value of 16 μM. Both ammonia and glutamine inhibited N2 fixation (C2H2 reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.

Regulation of nitrogen fixation by nitrite and glutamine inDerxia gummosa

Nitrogenase activity of cells of Derxia gummosa (30 h growth in cultures without combined nitrogen) was not inhibited on adding nitrate. However, on adding either azaserine or methionine sulfoximine (MSX) with nitrate to these cells, nitrogenase (C2H2 reduction) was inhibited because nitrite accumulated in the reaction mixtures. Nitrite inhibition of the in vivo C2H2 reduction had a Ki value of 16 μM. Both ammonia and glutamine inhibited N2 fixation (C2H2 reduction) in intact cells and in those treated with toluene. This inhibition by ammonia was relieved by methionine sulfoximine but not by glutamine. Azaserine enhanced the inhibition of nitrogenase produced by either ammonia or glutamine, since these treatments resulted in an accumulation of glutamine.

The catalytic activity of nitrogenase in intact Azotobacter vinelandii cells

The influence of the growth conditions on the concentration of nitrogenase and on the nitrogenase activity, was studied in intact Azotobacter vinelandii cells. It was observed that whole cell nitrogenase activity could be enhanced in two ways. An increase of the growth rate of cells was accompanied by an increase in whole cell nitrogenase activity and by an increase in the concentration of nitrogenase in the cells. The molar ratio of Fe protein:MoFe protein was 1.47 +/- 0.17 and independent of the growth rate. Activity measurements in cell extracts showed that the catalytic activity of the nitrogenase proteins was independent of the growth rate of cells. The second way to increase whole cell nitrogenase activity was to expose cells to excess oxygen. Whole cells were exposed for 2.5 h to an enhanced oxygen-input rate. After this incubation nitrogenase activity was increased without an increase in protein concentration. It is calculated that the catalytic activity of the Fe protein in these cells was 6200 nmol C2H4 formed X min-1 X (mg Fe protein)-1. With these cells and with cells grown at a high growth rate, 50% of the whole cell activity is lost by preparing a cell-free extract. It will be demonstrated that this inactivation is partly caused by the activity measurements in vitro. When dithionite was replaced by flavodoxin as electron donor, a maximal catalytic activity of 4500 nmol C2H4 formed X min-1 X (mg Fe protein)-1 was measured in vitro for the Fe protein. The results are discussed in relation to the present model for nitrogenase catalysis.

Electron allocation to H+ and N2 by nitrogenase in Rhizobium leguminosarum bacteroids.

Electron allocation to H+ and N2 by nitrogenase in intact Rhizobium leguminosarum bacteroids has been studied. Nitrogenase activity was measured in intact cells with succinate and oxygen substrates. When whole cell nitrogenase activity was inhibited by oxygen-limitation or by the addition of the H+-conducting ionophore carbonylcyanide m-chlorophenylhydrazone, both inducing a low intracellular ATP/ADP ratio, the electron allocation to H+ was favoured over that to N2. When whole cell nitrogenase activity was inhibited by excess oxygen or by the addition of the K+-conducting ionophore valinomycin, both inhibiting electron transport to nitrogenase without affecting the intracellular ATP/ADP ratio, no effect upon the electron allocation to H+ and N2 was observed. The whole cell experiments could be confirmed by experiments with bacteroids treated with hexadecyltrimethylammonium bromide. Nitrogenase is highly active in these preparations with Na2S2O4 and MgATP as substrates. No effect was observed upon electron allocation to H+ and N2 when nitrogenase was inhibited by limitation of reductant (Na2S2O4) or MgATP. Only when nitrogenase was inhibited by MgADP, electron allocation to H+ was favoured. The amount of nitrogenase component 1 and 2 in bacteroids was estimated with protein blotting, followed by an immunological detection. It was found that 17% +/- 3% of total bacteroid protein is component 1 and 12% +/- 2% is component 2. The specific nitrogenase activity of bacteroids treated with hexadecyltrimethylammonium bromide is 178 +/- 62 nmol C2H4 formed X min-1 X mg total protein-1. Despite the high protein concentrations, nitrogenase is not inhibited. With cell-free extracts or with purified nitrogenase components isolated from R. leguminosarum bacteroids, electron allocation to H+ was favoured over that to N2, independently of the mechanism of inhibition. The discrepancies between the whole cell studies and those with isolated enzyme will be discussed with respect to the present mechanism of action of nitrogenase.

Short-term regulation of the nitrogenase activity in Rhodopseudomonas sphaeroides.

The nitrogenase activity in whole cells of Rhodopseudomonas sphaeroides could be inhibited by lowering the electrical potential across the cytoplasmic membrane. The membrane potential was partly dissipated either by lowering the light intensity or by the addition of a lipophilic cation, tetraphenylphosphonium. Under these circumstances, it was shown that the intracellular ATP/ADP ratio was not affected and that the inhibition of the whole cell nitrogenase activity was not due to an inactivation of the nitrogenase enzyme. From these results it is concluded that electron transport to nitrogenase in Rps. sphaeroides is dependent on a high membrane potential. The nitrogenase enzyme in whole cells could be inactivated by lowering the membrane potential across the cytoplasmic membrane by incubating the cells in the dark or in the light in the presence of uncouplers. Nitrogenase could be reactivated in the light in the absence of uncouplers. Some possible mechanisms of action of NH+4 inhibition of whole cell nitrogenase activity could be excluded. Inhibition by NH4Cl of whole cell nitrogenase activity in Rps. sphaeroides could neither be explained by a rapid inactivation of the nitrogenase enzyme, nor by an effect on the intracellular ATP/ADP ratio or the membrane potential. NH+4 inhibits whole cell nitrogenase activity not directly but probably after being assimilated by glutamine synthetase. The role of glutamine, glutamate and 2-oxoglutarate on the regulation of electron transport to nitrogenase will be discussed.